Use of coenzyme factor for activation of atp production in cell

ABSTRACT

Problem: to provide an activator for activating intracellular ATP production. Solution: use of a 5-deazaflavin compound represented by the following formula (I): 
     
       
         
         
             
             
         
       
     
     (wherein, R 1  represents a hydrogen atom, an alkyl group, a halogen-substituted alkyl group, a carboxy-substituted alkyl group, or a phenyl group, R 2  represents an alkyl group, a cycloalkyl group, a phenyl-substituted lower alkyl group, a phenyl group, a phenyl group substituted by one of a halogen atom, a lower alkyl group, or a lower alkoxy group, or a lower alkyl disubstituted phenyl group, and R 3  and R 4  each represent a hydrogen atom, a lower alkyl group, a halogen atom, a hydroxyl group, a nitro group, a cyano group, a lower alkoxy group, a phenyl-substituted lower alkoxy, a lower alkylamino group, a phenyl-substituted lower alkylamino group, or a lower alkylsulfonyl group).

TECHNICAL FIELD

The present invention relates to use of a coenzyme factor for activatingintracellular ATP production, more specifically, use of a coenzymefactor that assists a redox enzyme involved in ATP production.

BACKGROUND ART

Neurodegenerative diseases and depression that accompany Alzheimer'sdisease, Parkinson disease, or cerebral hemorrhage/infarction have showna drastic increase with the coming of an aging society in recent yearsand there is increasingly a demand for preventive/remedy of theseneurodegenerative diseases.

One of the onset factors of the above-described diseases is thedysfunction of energy production (ATP production) in cells. ATPproduction occurs in the cytoplasmic matrix, but occurs mainly in themitochondrial complex under aerobic conditions. Mitochondria are presentin all the animal cells and are involved in energy production necessaryfor the activities of cells via an electron transport system. Sincemitochondria and mitochondrial genes are vulnerable to oxidative stresscaused by abnormal electron transport, they will result in lower energymetabolism and cell degeneration. Particularly in brain cells requiringa high energy amount, mitochondria are more susceptible to denaturationso that so-called “brain mitochondrial dysfunctions” due tomitochondrial abnormal electron transport that accompanies aging iscaused.

On the other hand, a recent research suggests that retardation of agingand prolongation of the lifespan of organisms can be achieved by theactivation of Sirtuin genes (Non-Patent Document 1).

A study on the relation between sirtuin genes and mitochondria hasproceeded and it suggests that with the activation of sirtuin genes,mitochondria which are intracellular organelles increase in their amountand are activated further not only to promote prevention of dementia,prevention of arteriosclerosis, prevention of hearing impairment, fatcombustion, cellular repair, and removal of active oxygen and therebysuppress expression of aging factors but also to provide an effect forcuring mitochondrial dysfunctions (Non-Patent Document 2).

For example, the research team of The University of Massachusetts founda memory disorder from mice whose SIRT1 gene, one of sirtuin genes, hadbeen knocked out and advocated the possibility of the present geneparticipating in memory. Further, the team suggested application ofSIRT1 gene activation to the therapy of neurodegenerative diseases byusing animal models of Alzheimer's disease and amyotrophic lateralsclerosis. In 2008, on the other hand, Shin-ichiro Imai, a professor ofWashington University School of Medicine, identified NMN (Nicotinamidemononucleotide) as a substance controlling the human aging and alsosucceeded in use of NMN for the reactivation of islets of Langerhanswhich had once been inactivated with aging. It has been revealed thatNMN activates all the genes and vitalizes mitochondria (Non-PatentDocument 3).

It has also been revealed that SIRT2, one of longevity protein sirtuingenes discovered by Prof. Imai, et al., is a heterochromatin componentthat silences transcription at telomeres and the ribosomal DNA and is anNAD-dependent histone deacetylase. This enzyme serves to restore bindingof DNA to histones, which has once been weakened by acetylation, bydeacetylation, enhance wrapping of DNA around histones, and therebysuppress transcription, which is presumed to be associated withlongevity (Non-Patent Document 4).

Suggesting the possibility that a biosynthesis promoter of NAD⁺(Nicotinamide Adenine Dinucleotide), an activation factor of the enzyme,has an antiaging effect, Prof. Imai, et al. have reported thepossibility that NMN, an intermediate substance for NAD⁺ synthesis,promotes biosynthesis of NAD⁺ and this is associated with an antiagingeffect (Non-Patent Document 5).

Further, Mills, et al. have reported that long-term administration of adiet comprising NMN to mice enhances NAD⁺ production in the tissue andcan mitigate age-associated physiological dysfunction of individuals(Non-Patent Document 6).

This can be understood from the fact that NAD⁺ is one of the mostimportant molecules in energy production which is associated with thefirst stage of oxidative phosphorylation in mitochondria and anoxidation reaction in the TCA cycle.

It is known, on the other hand, that SIRT1, one of longevity proteinsirtuin genes, is mainly associated with repair/regeneration ofmitochondria that have been injured, though expressed in nuclei.Further, it has been revealed that SIRT3, SIRT4, and SIRT5, of sevenmammalian sirtuin genes that have already been discovered, are expressedmainly in mitochondria (Non-Patent Documents 7 and 8).

It is presumed comprehensively based on these results that varioussirtuin genes are associated with a lifespan extending effect viaactivation of mitochondrial function (Non-Patent Document 9).

In mitochondria when normal, NMN (a precursor compound of NAD⁺ (nicotineamide adenine dinucleotide) is always produced continuously, but whenaged or sick, its production amount decreases, leading to a decrease inNAD⁺. As a result, it causes a reduction in ATP (adenosinetriphosphate), a life support energy source produced by oxidativephosphorylation in mitochondria through an electron transport chain or aglycolytic pathway in the cytoplasmic matrix (Non-Patent Document 10).NADH (Dihydro-nicotinamide adeninedinucleotide), a reduced form of NAD⁺,or FADH₂ (Dihydro-flavin adenine dinucleotide) functions as a coenzymein the TCA (citric acid) circuit of the intramitochondrial matrix or thecytoplasmic glycolytic pathway.

NAD (nicotinamide adenine dinucleotide) is one of coenzymes associatedwith a redox enzyme and its reduced form, NADH, is a coenzyme mostabundantly present in the body.

NAD has a structure in which nicotinamide mononucleotide (NMN) andadenylic acid constitute a phosphodiester bond. Since the nitrogen atomof the pyridine ring in NAD, the oxidized form, is present as a pyridiumion, it is expressed as “NAD⁺”. The important function of NAD⁺ residesin that the reduction of NAD⁺ is conjugated with an ATP productionmechanism (oxidation reaction).

FAD (Flavin adenine dinucleotide) is a coenzyme also associated with aredox enzyme and FADH₂, a reduced form thereof, is used for ATPproduction in the mitochondrial electron transport chain.

A primary supply destination of FAD in the metabolism of eukaryoticorganisms is the intramitochondrial citric acid circuit and thecytoplasmic matrix where β oxidation is performed. In the citric acidcircuit, FAD functions as a coenzyme of succinate dehydrogenase thatoxidizes succinic acid into fumaric acid, while in the β oxidation, itfunctions as a coenzyme for the enzymatic reaction of acyl CoAdehydrogenase. It is however presumed to be difficult to develop NMN orNAD⁺ belonging to a redox (reduction oxidation) type as a pharmaceuticalin future, because it is easily decomposed in the metabolic system andin addition, is chemically instable. Moreover, synthesis of it costshigh.

There is therefore an eager demand for the development of a chemicalfully satisfactory as a pharmaceutical from the viewpoint that it has anexcellent effect for activating the ATP production function and showsexcellent pharmacokinetics (biological absorption, intracerebralmigration, and the like).

The present inventors have proceeded with an extensive investigation onthe synthesis of a compound having a flavin skeleton and apharmacological effect thereof for years. A 5-deazaflavin(pyrimido[4,5-b]quinoline-2,4(3H,10H)-dione) derivative obtained bysubstituting N at position 5 of riboflavin with CH and having a specialchemical structure is a compound synthesized for the first time as ariboflavin analog in 1970. Some of 5-deazaflavin derivatives are knownto show excellent antitumor activity or have anti-herpesvirus activityand some of them are used as an antitumor agent or an anti-herpesvirusagent (Patent Documents 1 to 5).

The derivatives have also been studied in terms of a catalytic functionfor a conversion reaction of an alcohol into ketone or aldehyde thatoccurs with the change of the structure of the compound, a conversionreaction from amine to imine (ketone), reduction of a carbonyl compoundinto an alcohol, reduction or asymmetric reduction of an imine to anamine, and similarly to NAD⁺ (nicotinamide nucleotide) (Non-PatentDocument 11). Various patent documents describing them as amitochondrial function activator have been published (Patent Documents 6to 8). A document paying attention to redox catalytic activity of the5-deazaflavin derivatives and applying it to a mitochondrial functionactivating effect has not yet been found. It is obvious that since5-deazaflavin derivatives have redox catalytic activity, they have aninfluence on the ATP production function in mitochondrial oxidativephosphorylation. The 5-deazaflavin skeleton is found in a coenzyme F₄₂₀(Coenzyme F₄₂₀) which is a coenzyme associated with a redox reaction ofmethane fermentation and it is also a flavin analog. Its redox behavioris more similar to that of NAD(P)⁺ than to that of flavin. One of theresonance canonical formulas of it includes, in the molecule thereof, aNAD(P)⁺ structure and it can be regarded as a “flavin type NAD(P)⁺” alsofrom the electron density (determined by molecular orbital computationalchemistry).

The present inventors have carried out an extensive investigation,considering that since 5-deazaflavins have a redox (oxidation andreduction) function similar to that of NAD⁺ or FAD, they activateintracellular ATP production and further, directly or indirectlyactivate sirtuin genes. As a result, it has been found that a5-deazaflavin compound having a certain structural formula is verychemically stable, can be synthesized at a low cost, activates ATPproduction, and activates sirtuin genes that control the mitochondrialfunction, leading to the completion of the present invention.

CITATION LIST Patent Documents

Patent Document 1: Japanese Patent No. 3073309

Patent Document 2: Japanese Patent Application Laid-Open No. H6-199857

Patent Document 3: Japanese Patent Application Laid-Open No. H11-322746

Patent Document 4: Japanese Patent Application Laid-Open No. 2000-212087

Patent Document 5: Japanese Patent Application Laid-Open No. H6-73058

Patent Document 6: Japanese Patent Application Laid-Open No. 2001-48784

Patent Document 7: Japanese Patent Application Laid-Open No. 2002-322058

Patent Document 8: Japanese Patent Application Laid-Open No. 2008-255059

Non-Patent Documents

Non-Patent Document 1: 140. Mitochondrial control for antiaging,Research reports of The Uehara Memorial Foundation, 25 (2011)

Non-Patent Document 2: Elucidation of metabolic control mechanism ofSIRT1, www.mishima-kaiun.or.jp/assist/docs/SNo4-hasegawa.pdf

Non-Patent Document 3: Body function recovery and prolongation ofhealthy lifespan—Unexpected effect of a substance “NMN” clarified by theresearch of aging and lifespan,https://www.mugendai-web.jp/archives/6576

Non-Patent Document 4: Imai S1, Armstrong CM, Kaeberlein M, GuarenteL(2000), Transcriptional silencing and longevity protein Sir2 is anNAD-dependent histone deacetylase. Nature. 403(6771):795-800.

Non-Patent Document 5: Imai (2010) A possibility of nutriceuticals as ananti-aging intervention: activation of sirtuins by promoting mammalianNAD biosynthesis. Pharmacol Res. 62(1):42-7.

Non-Patent Document 6: Mills K F, Yoshida S, Stein L R, Grozio A, KubotaS, Sasaki Y, Redpath P, Migaud M E, Apte R S, Uchida K, Yoshino J, ImaiS I (2016) Long-Term Administration of Nicotinamide MononucleotideMitigates Age-Associated Physiological Declinde in Mice. Cell Metab.24(6):795-806.

Non-Patent Document 7: Michishita E1, Park J Y, Burneskis J M, Barrett JC, Horikawa I. (2005) Evolutionarily conserved and nonconserved cellularlocalizations and functions of human SIRT proteins. Mol Biol Cell.October; 16(10):4623-35

Non-Patent Document 8: Pacholec M, Bleasdale J E, Chrunyk B, CunninghamD, Flynn D, Garofalo R S, Griffith D, Griffor M, Loulakis P, Pabst B,Qiu X, Stockman B, Thanabal V, Varghese A, Ward J, Withka J, Ahn K.(2010) SRT1720, SRT2183, SRT1460, and resveratrol are not directactivators of SIRT1. J Biol Chem. 285(11):8340-51.

Non-Patent Document 9: Tang BL(2016) Sirt1 and the Mitochondria Mol.Cells 39(2):87-95

Non-Patent Document 10: The pathophysiological importance andtherapeutic potential of NAD⁺ biosynthesis in age-related diseases,https://seikagaku.jbsoc.or.jp/10.14952/SEIKAGAKU.2015.870239/data/index.pdf

Non-Patent Document 11: “Synthesis of heterocyclic compound having redoxfunctionality”,www.waseda.jp/prj-prac-chem/product/research%20product/2002 nitta.pdf

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a 5-deazaflavincompound selected from various 5-deazaflavin derivatives and having astructural formula effective for activating intracellular ATPproduction.

Means for Solving the Problems

With a view to achieving the above-described object, the presentinventors carried out an extensive investigation, prepared various5-deazaflavin derivative compounds having respectively differentstructural formulas as a specimen, and repeated various experiments invitro with those compounds. As a result, they have found a compoundhaving a structural formula capable of increasing intracellular ATPproduction.

First, the present inventors payed attention to, as an analog of NMN(refer to FIG. 17), 5-deazaflavin obtained by substituting, by amethylene group, nitrogen at position 5 of the flavin skeleton ofriboflavin (Vitamin B₂) known as a growth factor and being awater-soluble vitamin. 5-Deazariboflavin was metabolically antagonisticto riboflavin and showed strong anti-coccidium activity. A coenzyme F₄₂₀(refer to FIG. 18) having a 5-deazaflavin skeleton has recently beenfound from a methanogen and known to have an important role in thereduction process of a carbon dioxide gas into methane or thebiosynthesis process of an antibiotic. The redox behavior of5-deazaflavin is similar to that of NAD(P)⁺ (refer to FIG. 19) ratherthan that of flavin. One of the resonance canonical formulas includes,in the molecule thereof, a NAD(P)⁺ structure and it can be regarded as a“flavin type NAD(P)⁺” also from the electron density (determined bymolecular orbital computational chemistry) (refer to FIG. 20). Huckel MOcalculation has revealed that a 5-deazaflavin ring is much deficient, atposition 5 thereof, in n electrons (net charge: +0.24) and alsonicotinamide nucleotide is similarly deficient, at position 4 thereof,in n electrons (net charge: +0.210). The 5-deazaflavin ring is thereforepresumed to belong to both NAD(+) and FAD (refer to FIG. 21) electrontransport chains. Nicotinamide adenine dinucleotide (NAD) is an electrontransporter used in all the eukaryotic organisms and manyarchaebacterial and eubacteria.

In fact, fluorescent labeling for mitochondrial membrane potential byMitoTracker fluorescent staining showed that treatment with a novel NMNanalog (5-deazaflavin) markedly promoted mitochondrial activity. It ispresumed that activation of sirtuin genes activates intracellularorganelles “mitochondria”, promotes prevention of dementia, preventionof arteriosclerosis, prevention of hearing impairment, fat combustion,cellular repair, and removal of active oxygen harmful for genes, andthereby has an effect for suppressing the expression of aging factors.Compared with β-NMN, 5-deazaflavin serving as an electron transportchains switches on and activates sirtuin genes which are longevity genesat a low dose and at the same time, promotes ATP production by theactivation of mitochondria.

The present inventors have presumed that compared with β-NMN whose useis under investigation as a pharmaceutical capable of supplementing NMN,this 5-deazaflavin compound having a redox function similar to that ofNAD⁺ or FAD is very chemically stable and can be synthesized at a lowcost, activates NAD⁺, and directly or indirectly activates the longevitygenes (SIRT1 and SIRT3) of mitochondria. In other words, for the purposeof evaluating the activation of the longevity gene SIRT1 which is NAD⁺dependently activated and has already been known as a gene important forthe maintenance of mitochondrial function, the present inventors carriedout screening evaluation of an mRNA increase via q-RT-PCR using HCT116cells, with the expression of FOX01, a target transcription factor ofSIRT1, as an activity index.

As a result, they have revealed that compared with β-NMN, the compoundsof the present invention have a SIRT1 activating ability at a less dose.The compounds of the present invention are therefore expected todirectly activate NAD⁺, produce ATP continuously even under pathologicconditions such as ischemia, and thus have a continuous and powerfuleffect. The compounds of the present invention are therefore presumed tosufficiently have necessary conditions for the development of them as apharmaceutical. In addition, they have a chemical structure close to anintegrated structure of the core portions of the coenzymes NAD⁺ and FADfrom the viewpoints of function and electron theory, in other words,close to a structure present in the natural world as a hybrid ofnicotinamide and flavin, so that they are presumed to produce almost noside effects. Further, the structure of the compounds of the presentinvention have a redox potential higher than that of NAD or FAD as acoenzyme and are excellent in redox ability judging from the measuredvalue of their redox potential.

The present invention has been completed based on the above findings.

Described specifically, the present invention identifies compoundsrepresented by the following formulas (I) to (IV) as a coenzyme factoreffective for activating intracellular ATP production.

(wherein, R₁ represents a hydrogen atom, an alkyl group, ahalogen-substituted alkyl group, a carboxy-substituted alkyl group, or aphenyl group, R₂ represents an alkyl group, a cycloalkyl group, aphenyl-substituted lower alkyl group, a phenyl group, a phenyl groupsubstituted by one of a halogen atom, a lower alkyl group, or a loweralkoxy group, or a lower alkyl disubstituted phenyl group, and R₃ and R₄each represent a hydrogen atom, a lower alkyl group, a halogen atom, ahydroxyl group, a nitro group, a cyano group, a lower alkoxy group, aphenyl-substituted lower alkoxy, a lower alkylamino group, aphenyl-substituted lower alkylamino group, or a lower alkylsulfonylgroup).

(wherein, R₁ and R₃ each represent a hydrogen atom, an alkyl group, ahalogen-substituted alkyl group, a carboxy-substituted alkyl group, aphenyl group, a phenyl group substituted by one of a halogen atom, alower alkyl group, or a lower alkoxy group, or a lower alkyldisubstituted phenyl group and R₂ represents an alkyl group, acycloalkyl group, a phenyl-substituted lower alkyl group, a phenylgroup, a phenyl group substituted by one of a halogen atom, a loweralkyl group, or a lower alkoxy group, or a lower alkyl disubstitutedphenyl group).

(wherein, R₁ represents a hydrogen atom, an alkyl group, ahalogen-substituted alkyl group, a carboxy-substituted alkyl group, aphenyl group, or a phenyl group substituted by one of a halogen atom, alower alkyl group, or a lower alkoxy group and R₂ represents an alkylgroup, a cycloalkyl group, a phenyl-substituted lower alkyl group, aphenyl group, a phenyl group substituted by one of a halogen atom, alower alkyl group, or a lower alkoxy group, or a lower alkyldisubstituted phenyl group).

(wherein, R₁ represents a hydrogen atom, an alkyl group, ahalogen-substituted alkyl group, a carboxy-substituted alkyl group, aphenyl group, or a phenyl group substituted by one of a halogen atom, alower alkyl group, or a lower alkoxy group and R₂ represents an alkylgroup, a cycloalkyl group, a phenyl-substituted lower alkyl group, aphenyl group, a phenyl group substituted by one of a halogen atom, alower alkyl group, or a lower alkoxy group, or a lower alkyldisubstituted phenyl group).

Advantageous Effect of the Invention

Using a coenzyme factor provided by the present invention and capable ofactivating ATP production can improve the dysfunction of energyproduction in cells.

It is therefore extremely useful as a preventive/remedy forneurodegenerative diseases and depression that accompany Alzheimer'sdiseases, Parkinson's diseases, cerebral hemorrhage/infarction, or thelike.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the fluorescence measurement results of intracellular andextracellular ATP concentrations of cultured cells of a human-derivedneuroblast (neuroblastoma) SH-SYSY strain to which Specimen 1 was added.

FIG. 2A shows the fluorescence measurement results of culturedastrocytes, one of glial cells of the central nerve system, to whichSpecimen 1 was added.

FIG. 2B shows the quantified measurement results of FIG. 2A.

FIG. 3 shows relative comparison between the intracellular ATPconcentration after addition of Specimens 1 and 2 to cultured humanglioma U251 cells (Sigl-R transfected cells) and that without addition(Cont.).

FIG. 4 is an immunostained image showing elongation/growth/branching ofa neuron axon of a hippocampal neuron after collecting the hippocampalneuron from a juvenile (0 day after birth) ICR mouse brain, culturingthe neurons on a culture dish, and adding Specimen 1 thereto.

FIG. 5 shows the measurement results of the number of branches of ahippocampal neuron axon after Specimen 1 was added thereto as in FIG. 4.

FIG. 6 is an immunostained image showing elongation/growth/branching ofa hippocampal neuron dendrite after Specimen 1 was added thereto as inFIG. 4.

FIG. 7 shows the measurement results of the number of branches of ahippocampal neuron dendrite after Specimen 1 was added thereto as inFIG. 6.

FIG. 8 is an immunostained image of a synapse in an autapse preparationobtained by culturing as in FIG. 4 and immunostaining an excitatorysynapse on culture day 14.

FIG. 9 shows the number of excitatory synapses on culture day 14quantified after culturing as in FIG. 4.

FIG. 10 is an immunostained image of a hippocampal neuron dendritequantified three days (on culture day 14) after single administration ofSpecimen 1 to a mature cultured hippocampal neuron (on culture day 11).

FIG. 11 shows the number of branches of a dendrite measured afterimmunostaining with a MAP2 antibody in the growth stage on culture day14 as in FIG. 10.

FIG. 12 shows the measurement of a K⁺ depolarization-induced Ca²⁺concentration increase in mitochondria present in cerebral cortexneurons (neurons) and an inhibitory effect of Specimen 3 thereon. Thisdepolarization of the cellular membrane is caused by intraperitoneallyadministering physiological saline (Control) and Specimen 3 (10 μg/kg)to adult C57BL/6N mice (from 10 to 17 week old, weight: from 28 to 31g), forming a thin brain slice including the cortex and the hippocampusregions after 22 hours, and adding 80 mM KCl (80 K) to an extracellularfluid.

FIG. 13 shows, similar to FIG. 12, the measurement of a K⁺depolarization-induced Ca²⁺ concentration increase in mitochondria inhippocampal neurons (neurons) and an inhibitory effect of Specimen 3thereon.

FIG. 14 shows the respective brain cross-sections of intracerebralhemorrhage model rats obtained by making a small hole into the skull ofadult Wistar rats (weight: from 200 to 230 g) under inhalationanesthesia and then injecting 1.2 μl of a physiological saline to theright brain striatum in Control group and a physiological salinecomprising 0.24U collagenase (type N) to that in a hemorrhage group.

FIG. 15 shows the quantitative measurement of exercise footprints of anintracerebral hemorrhage model rat after injecting 100 μg/kg of Specimen4 to the brain spot thereof one hour after collagenase administration inorder to verify the brain cell protective action of Specimen 4.

FIG. 16 shows the daily changes of an exercise distance and an exercisespeed of a rat administered or not administered with Specimen 4, whichwas measured as in FIG. 15.

FIG. 17 shows the chemical structural formula of β-NMN.

FIG. 18 shows the chemical structural formula of a coenzyme F₄₂₀.

FIG. 19 shows the chemical structural formula of NAD(P)⁺ and describesthe redox reaction thereof.

FIG. 20 shows electron densities determined by molecular orbitalcomputational chemistry of the flavin rings of 5-deazaflavin andNAD(P)⁺.

FIG. 21 is a view describing the similarity of the structure betweenNAD⁺ and FAD.

FIG. 22 shows the quantification and test results of the number ofbranches of the hippocampal neuron dendrite after addition of Specimen5.

FIG. 23 shows the quantification and test results of the number ofbranches of the hippocampal neuron dendrite after addition of Specimen6.

FIG. 24 shows the quantification and test results of the number ofbranches of the hippocampal neuron dendrite after addition of Specimen7.

FIG. 25 shows the quantification and test results of the number ofbranches of the hippocampal neuron dendrite after addition of Specimen8.

FIG. 26 shows the quantification and test results of the number ofbranches of the hippocampal neuron dendrite after addition of Specimen9.

FIG. 27 shows the quantification and test results of the number ofbranches of the hippocampal neuron dendrite after addition of Specimen10.

FIG. 28 shows the quantification and test results of the number ofbranches of the hippocampal neuron dendrite after addition of Specimen10.

FIG. 29 shows the analysis results of the mitochondrial Ca²⁺ of thebrain preparations of mice pretreated for 24 hours with β-NMN bysubcutaneous administration. Preparations stained with Xrhod-1, amitochondria-selective Ca²⁺ indicator, were subjected to an operationincluding 80K ACFS exposure for 5 minutes and washing for 5 minutesthree times repeatedly. The dose response relationship of β-NMNeffecting the mitochondrial Ca²⁺ variations at that time: A: Control, B:10 mg/kg of β-NMN, C: 30 mg/kg of β-NMN, and D: 100 mg/kg of β-NMN.β-NMN was subcutaneously administered at each dose and whole brain slicepreparations were formed after 24 hours and stained with Xrhod-1, amitochondria-specific Ca²⁺ indicator. The time-dependent increase inmitochondrial Ca²⁺ obtained by three times stimulation with 80K ACSF wasmeasured from the red fluorescence imaging (>600 nm) when excited at 580nm. Solid line: response at a cerebral cortex (CTX) site; dotted line:response at a hippocampal CA1 site (CA1). Shown are an average of from 5to 6 cases and a standard deviation.

FIG. 30 shows the analysis results of the mitochondrial Ca²⁺ of brainpreparations of mice pretreated for 24 hours with Specimen 1 (TND1128)by subcutaneous administration. Preparations stained with Xrhod-1, amitochondria-selective Ca²⁺ indicator, were subjected to an operationincluding 80K ACFS exposure for 5 minutes and washing for 5 minutesthree times repeatedly, as in FIG. 29. The dose response relationship ofSpecimen 1 (TND1128) effecting the mitochondrial Ca²⁺ variations at thattime A: Control, B: 0.01 mg/kg of TND1128, C: 0.1 mg/kg of TND1128, andD: 1.0 mg/kg of TND1128 (subcutaneous administration). As in FIG. 29,the time-dependent mitochondrial Ca²⁺ increase obtained by three timesstimulation with 80K ACSF was measured from the red fluorescence imaging(>600 nm) of Xrhod-1 loaded preparations when excited at 580 nm. Solidline: response at a cerebral cortex (CTX) site; dotted line: response ata hippocampal CA1 site (CA1). Shown are an average of from 5 to 6 casesand a standard deviation.

FIG. 31 shows the dose response relationship of the protective effectsof β-NMN on a mitochondrial Ca²⁺ concentration when exposed to 80K ACSF.The data in FIG. 29 is quantitatively shown (refer to FIG. 37). A:effect of β-NMN on the total mitochondrial Ca²⁺ uptake amount at thecerebral cortex (CTX) (white) and the hippocampus (CA1) (black) whenexposed to 80K ACSF three successive times. B:

the dose response relationship of the mitochondrial Ca²⁺ uptake eachtime when exposed to 80K ACSF at the cerebral cortex. C: the doseresponse relationship of the mitochondrial Ca²⁺ uptake each time whenexposed to 80K ACSF at the hippocampus. *: Significant difference(P<0.05) provided by multiple comparison by Tukey's method.

FIG. 32 shows the dose response relationship of the protective effectsof Specimen 1 (TND1128) on a mitochondrial Ca²⁺ concentration whenexposed to 80K ACSF. The data in FIG. 29 is quantitatively shown (referto FIG. 37). A: effect of TND1128 on the total mitochondrial Ca²⁺ uptakeamount at the cerebral cortex (CTX) (white) and the hippocampus (CA1)(black) when exposed to 80K ACSF three successive times. B: the doseresponse relationship on the mitochondrial Ca²⁺ uptake each time whenexposed to 80K ACSF in the cerebral cortex. C: the dose responserelationship on the mitochondrial Ca²⁺ uptake each time when exposed to80K ACSF in the hippocampus. *: Significant difference (P<0.05) providedby multiple comparison by Tukey's method.

FIG. 33 shows the analysis results of the cytoplasmic Ca²⁺ of the micebrain preparations pretreated for 24 hours with β-NMN by subcutaneousadministration. β-NMN was subcutaneously administered at each dose,whole brain slice preparations were formed after 24 hours of theadministration, and the brain slice preparations were loaded with a Ca²⁺indicator Fura-4F capable of being retained in the cytoplasm. Atime-dependent cytoplasmic Ca²⁺ increase induced by three times 80K-ACSFstimulation obtained by exposing the brain preparations to excitedlights of 360 nm and 380 nm and finding a ratio of fluorescenceintensity (F360 and F380) of a bluish green color (>500 nm) at every 10seconds with the passage of time. A: Control, B: 10 mg/kg of β-NMN, C:30 mg/kg of β-NMN, and D: 100 mg/kg of β-NMN. Solid line: response at acerebral cortex (CTX) site; dotted line: response at a hippocampus CA1site (CA1). Shown are an average of from 5 to 6 cases and a standarddeviation.

FIG. 34 shows the analysis results of the cytoplasmic Ca²⁺ of the micebrain preparations pretreated for 24 hours with Specimen 1 (TND1128) bysubcutaneous administration. Preparations loaded with Fura-4F, acytoplasm-selective Ca²+ indicator, were subjected to an operationincluding 80K AFCS exposure for 5 minutes and washing for 5 minutesthree times repeatedly, as in FIG. 29. The dose response relationship ofTND1128 effecting the cytoplasmic Ca²⁺ variations at that time: A:Control, B: 0.01 mg/kg of TND1128, C: 0.1 mg/kg of TND1128, and D: 1.0mg/kg of TND1128 (subcutaneous administration). As in FIG. 33, a ratioof fluorescence intensity (F360 and F380) of a bluish green color (>500nm) of Fura-4F when the preparations were exposed to excited lights of360 nm and 380 nm was found at every 10 seconds with the passage oftime. Solid line: response at a cerebral cortex (CTX) site; dotted line:response at a hippocampus CA1 site (CAl). Shown are an average of from 5to 6 cases and a standard deviation.

FIG. 35 shows the dose response relationship of the protective effectsof β-NMN on the cytoplasmic Ca²⁺ concentration when exposed to 80K ACSF.The data in FIG. 33 are quantitatively shown (refer to FIG. 37). A:effects of β-NMN on the total cytoplasmic Ca²⁺ uptake amount at thecerebral cortex (CTX) (white) and the hippocampus (CA1) (black) whenexposed to 80K ACSF three successive times. B: the dose responserelationship of a cytoplasmic Ca²⁺ uptake each time when exposed to 80KACSF at the cerebral cortex. C: the dose response relationship of acytoplasmic Ca²⁺ uptake each time when exposed to 80K ACSF at thehippocampus.

FIG. 36 shows the dose response relationship of the protective effectsof Specimen 1 (TND1128) on the cytoplasmic Ca²⁺ concentration whenexposed to 80K ACSF. The data in FIG. 34 are quantitatively shown (referto FIG. 37). A: effects of β-NMN on the total cytoplasmic Ca²⁺ uptakeamount at the cerebral cortex (CTX) (white) and the hippocampus (CA1)(black) when exposed to 80K ACSF three successive times. B: the doseresponse relationship of the cytoplasmic Ca²⁺ uptake each time whenexposed to 80K ACSF at the cerebral cortex. C: the dose responserelationship of the cytoplasmic Ca²⁺ uptake each time when exposed to80K ACSF at the hippocampus. *Significant difference (P<0.05) providedby multiple comparison by Tukey's method.

FIG. 37 is a drawing for describing a quantification method of amitochondrial or cytoplasmic Ca²⁺ increase amount when exposed to 80Kthree successive times.

MODE FOR CARRYING OUT THE INVENTION

(Structure of Compound)

In the present invention, use of respective compounds having structuralformulas represented by the formulas (I), (II), (III), and (IV) iseffective for activating the intracellular ATP production.

Specific examples of these compounds and documents showing theproduction methods thereof are shown in Tables 1 to 4.

TABLE 1 (1)

Document of Compound production No R₁ R₂ R₃ R⁴ method I-1 H Me H H (4)I-2 H Me Me Me (4) I-3 H Et H H (4) I-4 H Et Me H (4) I-5 H n-Pr H H (4)I-6 H n-Bu H H (1) (4) I-7 H n-Bu NO₂ H (1) I-8 H n-Bu H Cl (1) I-9 HCH₃[CH₂]₇ H H (1) I-10 H CH₃[CH₂]₇ NO₂ H (1) I-11 H CH₃[CH₂]₇ H Cl (1)I-12 H CH₃[CH₂]₇ H OCH₂Ph (13) I-13 H CH₃[CH₂]₇ H OMe (13) I-14 HCH₃[CH₂]₇ H COMe (13) I-15 H CH₃[CH₂]₁₁ H H (1) I-16 H CH₃[CH₂]₁₁ NO₂ H(1) I-17 H CH₃[CH₂]₁₁ H Cl (1) I-18 H HO[CHOH]₃CH₂ H OBn (13) I-19 HHO[CHOAc]₃CH₂ H OBn (13) I-20 H Ph H H (1) I-21 H Ph NO₂ H (1) I-22 H PhH Cl (1) I-23 H Ph H OH (1) I-24 H 2,4-Me₂-C₆H₃ H H (1) I-25 H2,4-Me₂-C₆H₃ NO₂ H (1) I-26 H 2,4-Me₂-C₆H₃ H Cl (1) I-27 H 3,4-Me₂-C₆H₃H H (1) I-28 H 3,4-Me₂-C₆H₃ NO₂ H (1) I-29 H 3,4-Me₂-C₆H₃ H Cl (1) I-30H 4-Cl-C₆H₄ H H (1) I-31 H 4-Cl-C₆H₄ H Cl (1) I-32 Me Me H H (4) (9)I-33 Me Me Me H (6) I-34 Me Me H Me (5) I-35 Me Me H F (9) I-36 Me Me ClH (6) (9) I-37 Me Me H Cl (5) (9) I-38 Me Me Cl Cl (5) (9) I-39 Me Me HOH (1) (9) I-40 Me Me H OMe (5) (9) I-41 Me Me H N₃ (10) I-42 Me Me HNH₂ (10) I-43 Me Me H NMe₂ (10) I-44 Me Me H CN (6) (9) I-45 Me Me HNHCH₂Ph (9) I-46 Me Me H NHCOMe (10) I-47 Me Me H NHCOPh (10) I-48 Me MeH OCH₂Ph (9) I-49 Me Me -O-CH₂-O- (6) I-50 Me Et H H (4)Specimen 1 I-51Me Et Me H (4) I-52 Me Et H OH (12) I-53 Me n-Pr H H (1) (4) I-54 Men-Bu H H (1)Specimen 2 I-55 Me n-Bu H OH (1) I-56 Me n-Bu H OH (12) I-57Me C₆H₁₁ H Cl (1) I-58 Me CH₃[CH₂]₆ H H (2) I-59 Me CH₃[CH₂]₇ H H (2)I-60 Me CH₃[CH₂]₇ H F (14) I-61 Me CH₃[CH₂]₇ H OH (12) I-62 Me CH₃[CH₂]₇H NH(CH₂)₆NH₂ (14) I-63 Me CH₃[CH₂]₁₁ H H (2) I-64 Me CH₃[CH₂]₁₁ H OH(12) I-65 Me CH₃[CH₂]₁₇ H H (2) I-66 Me PhCH₂ H H (1) I-67 Me HO₂C[CH₂]₃H OH (12) I-68 Me HO₂C[CH₂]₅ H OH (12) I-69 Me Ph H H (1) I-70 Me Ph MeH (7) I-71 Me Ph H Me (7) I-72 Me Ph OMe H (7) I-73 Me Ph H OMe (7) I-74Me Ph Cl H (7) I-75 Me Pb H Cl (1)Specimen 4 I-76 Me Ph H Br (3) I-77 Me3-Me-C₆H₄ H H (1) I-78 Me 4-Me-C₆H₄ H H (1) I-79 Me 4-Me-C₆H₄ Me H (3)I-80 Me 4-Me-C₆H₄ H Me (3) I-81 Me 4-Me-C₆H₄ Cl H (11) I-82 Me 4-Me-C₆H₄OH H (11) I-83 Me 4-Me-C₆H₄ NMe₂ H (11) I-84 Me 4-Me-C₆H₄ SMe H (11)I-85 Me 4-Me-C₆H₄ SO₂Me H (11) I-86 Me 3,4-Me₂-C₆H₃ H H (7) I-87 Me3,4-Me₂-C₆H₃ NO₂ H (1) I-88 Me 4-MeO-C₆H₄ H H (7) I-89 Me 4-F-C₆H₄ H H(7) I-90 Me 4-Cl-C₆H₄ H H (1) I-91 Me 4-Cl-C₆H₄ H Cl (1) I-92 Me4-Br-C₆H₄ H H (3) I-93 Br(CH₂)₆ CH₃[CH₂]₇ H H (14) I-94 N₃(CH₂)₆CH₃[CH₂]₇ H H (14) I-95 NH₂(CH₂)₆ CH₃[CH₂]₇ H H (14) I-96 Ph Me H H (8)I-97 Ph Me H OH (1) I-98 Ph Et H H (8) I-99 Ph Et H OH (12) I-100 Phn-Pr H H (8) I-101 Ph n-Bu H H (1) (8) I-102 Ph n-Bu NO₂ H (1) I-103 Phn-Bu H OH (12) I-104 Ph n-Bu H Cl (1) I-105 Ph CH₃[CH₂]₅ H H (2) I-106Ph CH₃[CH₂]₇ H H (1) I-107 Ph CH₃[CH₂]₇ H Cl (1) (2) I-108 Ph CH₃[CH₂]₇H OH (12) I-109 Ph CH₃[CH₂]₁₁ H H (1) (2) I-110 Ph CH₃[CH₂]₁₁ H Cl (1)I-111 Ph CH₃[CH₂]₁₂ H OH (12) I-112 Ph CH₃[CH₂]₁₇ H H (2) I-113 Ph Ph HH (1) I-114 Ph Ph H Cl (1) I-115 Ph 3,4-Me₂-C₆H₃ H H (1) I-116 Ph3,4-Me₂-C₆H₃ H Cl (1) I-117 Ph 4-Cl-C₆H₄ H H (1) I-118 Ph 4-Cl-C₆H₄ H Cl(1) Documents: (1) T. Nagamatsu, Y. Hashiguchi, and Y. Yoneda, J. Chem.Soc., Perkin Trans. 1, 561-565 (1984) (2) K. Kuroda, T. Nagamatsu, Y.Sakuma, and F. Yoneda, J. Heterocyclic Chem., 19, 929-931 (1982) (3) K.Kuroda, T, Nagamatsu, R. Yanada, and F. Yoneda, J. Chem. Soc., PerkinTrans. 1, 547-550 (1993) (4) F. Yoneda, Y. Sakuma, S. Mizumoto, and R.Ito, J. Chem. Soc., Perkin Trans. 1, 1805-1808 (1976) (5) K. Mori, K.Shinozuka, Y. Sakuma, and F. Yoneda, J. Chem. Soc., Chem. Comm., 764(1978) (6) F. Yoneda, K. Mori, and Y. Sakuma, J. Chem. Soc., PerkinTrans. 1, 978-981 (1980) (7) F. Yoneda, K. Tsukuda, K. Shinozuka, F.Hirayama, K. Uekama, and A. Koshiro, Chem. Pharm. Bull., 28, 3049-3056(1980) (8) F. Yoneda, K. Mori, M. Ono, Y. Kadokawa, E. Nagao, and H.Yamaguchi, Chem. Pharm. Bull, 28, 3514-3520 (1980) (9) F. Yoneda, K.Mori, S. Matsuo, Y. Kadokawa, and Y. Samuma, J. Chem. Soc., PerkinTrans. 1, 1836-1839 (1981) (10) F. Yoneda, K. Mori, Y. Sakuma, and A.Koshiro, J. Heterocyclic Chem., 19, 945-947 (1982) (11) K. Tanaka, T.Kimura, X. Chen, T. Kawamoto, and F. Yoneda, Chem. Pharm. Bull, 38,312-317 (1990) (12) R. Hirayama, M. Kawase, T. Kimachi, K. Tanaka, andF. Yoneda, J. Heterocyclic Chem., 26, 1255-1259 (1989) (13) T. Kimachi,K. Tanaka, and F. Yoneda, J. Heterocyclic Chem., 28, 439-443 (1991) (14)Y. Eikyu, Y. Nakamura, T. Akiyama, F. Yoneda, K. Tanaka, and K. Fuji,Chem. Pharm. Bull., 40, 291-293 (1992)

TABLE 2 (II)

Com- Document of pound production No. R₁ R₂ R₃ method II-1 H CH₃[CH₂]₇ H(15) II-2 H CH₃[CH₂]₁₁ H (15) II-3 H CH₃[CH₂]₁₇ H (15) II-4 H Me Me (16)II-5 H Et Me (16) II-6 H n-Bu Me (16) (19) II-7 H CH₃[CH₂]₇ Me (16) (19)II-8 H CH₃[CH₂]₁₁ Me (16) (19) II-9 H CH₃[CH₂]₁₇ Me (18) (19) II-10 HC₆H₅[CH₂]₂ Me (16) II-11 Me Me Me (15) (16) (17) II-12 Me Et Me (15)(17) Specimen 5 II-13 Me n-Pr Me (16) (17) II-14 Me n-Bu Me (15) (16)(17) II-15 Me CH₃[CH₂]₇ Me (15) (16) (17) II-16 Me CH₃[CH₂]₁₁ Me (15)(16) II-17 Me CH₃[CH₂]₁₇ Me (15) II-18 Me Ph[CH₂]₂ Me (15) II-19 Me PhMe (15) II-20 Me 4-Me-C₆H₄ Me (15) II-21 Me 4-Cl-C₆H₄ Me (15) II-22 Me4-Br-C₆H₄ Me (15) II-23 Me 4-MeO-C₆H₄ Me (15) II-24 Me 3,4-Me₂-C₆H₄ Me(15) II-25 Br(CH₂)₁₀ n-Bu Me (19) II-26 Br(CH₂)₁₀ CH₃[CH₂]₇ Me (19)II-27 Br(CH₂)₁₀ CH₃[CH₂]₁₁ Me (19) II-28 Br(CH₂)₁₀ CH₃[CH₂]₁₈ Me (19)II-29 Ph Ph Ph (15) II-30 Ph 4-Me-C₆H₄ Ph (15) II-31 Ph 4-Cl-C₆H₄ Ph(15) Specimen 6 II-32 Ph 3,4-Me₂-C₆H₄ Ph (15) Documents: (15) T.Nagamatsu, H. Yamato, M. Ono, S. Takarada, and F. Yoneda, J. Chem. Soc.,Perkin Trans. 1, 2101-2109 (1992) (16) T. Nagamatsu, Y. Sakuma, and Y.Yoneda, Synthesis, 923-924 (1983) (17) F. Yoneda, T. Nagamatsu, M.Takamoto, Chem. Pharm. Bull, 31, 344-347 (1983) (18) F. Yoneda, H.Yamato, T. Nagamatsu, and H. Egawa, J. Polymer Sci.:Plymer Lett. Ed.,20, 667-670 (1982) (19) F. Yoneda, K. Tanaka, H. Yamato, K. Moriyama,and T. Nagamatsu, J. Am. Chem. Soc., 111, 9199-9202 (1989)

TABLE 3 (III)

Compound Document of No. R₁ R₂ production method III-1 H Me (20) (21)III-2 Me Me (20) (21) Specimen 7 III-3 Me Et (Unknown compound) III-4 Men-Bu (Unknown compound) Specimen 3 III-5 Me Bn (Unknown compound) III-6Me Ph (Unknown compound) III-7 Me 4-Me-C₆H₄ (Unknown compound) III-8 Me4-MeO-C₆H₄ (Unknown compound) III-9 Me 3,4-OCH₂O-C₆H₃ (Unknown compound)III-10 Me 4-HO-C₆H₄ (Unknown compound) III-11 Me 4-F-C₆H₄ (Unknowncompound) III-12 Me 4-Cl-C₆H₄ (Unknown compound) III-13 Me 4-Br-C₆H₄(Unknown compound) III-14 Ph Et (Unknown compound) Specimen 8 III-15 Phn-Bu (Unknown compound) III-16 Ph Bn (Unknown compound) III-17 Ph Ph(Unknown compound) III-18 Ph 4-Me-C₆H₄ (Unknown compound) III-19 Ph3,4-Me₂-C₆H₃ (Unknown compound) III-20 Ph 4-MeO-C₆H₄ (Unknown compound)III-21 Ph 4-F-C₆H₄ (Unknown compound) III-22 Ph 4-Cl-C₆H₄ (Unknowncompound) III-23 Ph 4-Br-C₆H₄ (Unknown compound) Documents: (20) T.Nagamatsu, H. Yamada, and K. Shiromoto, Heterocycles, 63, 9-16 (2004)(21) Japanese Patent Application Laid-Open No. 2005-104868

TABLE 4 (IV)

Compound No. R₁ R₂ Document of production method IV-1 H Me (22) IV-2 HEt (22) IV-3 H n-Bu (22) IV-4 H Ph (22) IV-5 H 3-Me-C₆H₄ (22) IV-6 H3,4-Me₂-C₆H₃ (22) IV-7 H 4-MeO-C₆H₄ (22) IV-8 H 4-F-C₆H₄ (22) IV-9 H4-Cl-C₆H₄ (22) IV-10 Me Me (22) Specimen 9 IV-11 Me Et (22) IV-12 Men-Bu (22) IV-13 Me Ph (22) IV-14 Me 3-Me-C₆H₄ (22) IV-15 Me 3,4-Me₂-C₆H₃(22) Specimen 10 IV-16 Me 4-MeO-C₆H₄ (22) IV-17 Me 4-F-C₆H₄ (22) IV-18Me 4-Cl-C₆H₄ (22) IV-19 Me 4-Br-C₆H₄ (22) IV-20 Ph Me (22) IV-21 Ph Ph(22) IV-22 Ph 3-Me-C₆H₄ (22) IV-23 Ph 3,4-Me₂-C₆H₃ (22) Specimen 11IV-24 Ph 4-MeO-C₆H₄ (22) IV-25 Ph 4-F-C₆H₄ (22) IV-26 Ph 4-Cl-C₆H₄ (22)Documents: (22) A. R. Shrestha, T. Shindo, N. Ashida, and T. Nagamatsu,Bioorg. & Med. Chem., 16, 8685-8696 (2008)

(Production Method of Compound)

D-deazaflavin compounds (from I-1 to I-118) (3) represented by theformula I can be synthesized by the production methods described in theknown documents (from 1 to 14). In particular, most of the derivativescan be synthesized by the general production method of Document (1)described in (Production method A). Described specifically, a6-N-substituted-aminourasil (1) and a suitable o-halogenobenzaldehyde(2) are heated and refluxed in dimethylformamide (DMF). The heating timefor from 3 to 7 hours is adequate. The reaction liquid is concentratedunder reduced pressure and a residue is recrystallized from a suitablesolvent (alcohol, dioxane, DMF, or the like) to obtain a corresponding5-deazaflavin (3).

Pyridodipyrimidine compounds (II) (from II-1 to II-32) (5) representedby the formula II can be synthesized by the production method describedin the known documents (from 15 to 19). In particular, most of thederivatives can be synthesized by the general production method ofDocument (15) described in (Production method B). Describedspecifically, a 6-N-substituted aminouracil (1) and a suitable3-substituted-6-chlorouraci-5-carbaldehyde (4) are heated and refluxedin dimethylformamide or acetic acid. The heating time for from 2 to 5hours is adequate. The reaction liquid is concentrated under reducedpressure and the residue is recrystallized from a suitable solvent(alcohol, acetic acid, DMF, or the like) to obtain a correspondingpyridodipyrimidine (5).

Deazaflavino-testosterone compounds (III) (from III-1 to III-2) (7)represented by the formula III can be synthesized by the productionmethod described in the known documents (20, 21). In a similar manner,the unknown compounds (from III-3 to III-23) can be synthesized.Described specifically, a 6-N-monosubstituted-aminouracil (1) and2-hydroxymethylene testosterone (6) are added to diphenyl ether. Afterfurther addition of p-toluenesulfonic acid, the resulting mixture isheated and stirred at 180° C. for from 30 minutes to 60 minutes in anargon atmosphere. After the reaction, the resulting reaction product issubjected to column chromatography to purify it. The reaction for thesynthesis can also be performed by heating under pressure in dioxane forseveral hours.

PRODUCTION EXAMPLE 1 (General synthesis of8′-substituted-5′-deaza-17β-hydroxy-3′-methylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dionederivatives (from III-3 to III-13))

To dioxane (50 ml) are added p-toluenesulfonic acid (60 mg, 0.32 mmol)and 6-(monosubstituted-amino)-3-methyluracil (1) (2.84 mmol), followedby the further addition of 2-hydroxymethylene testosterone (6) (1.0 g,3.16 mmol). Then, the resulting mixture is heated in a sealed tube for12 hours in argon atmosphere. After the reaction, the reaction productis separated and purified by column chromatography (Fuji Silysia, from230 to 400 mesh; eluent: ethyl acetate:ethanol=12:1 or ethyl acetatealone) to obtain crystalline powder. Further, recrystallization can becarried out using a mixed solution of ethyl acetate and n-hexane.

Compound III-3(5′-Deaza-8′-ethyl-17β-hydroxy-3′-methylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);yellow crystalline powder(0.77 g, 60%), mp 260° C. (decomp.); ¹H-NMR(300 MHz, CDCl₃) δ: 0.81 (3H, s, 18-CH₃), 1.00 (3H, s, 19-CH₃) , 1.41(3H, t, J=7.2 Hz, 8′-CH₂CH₃), 2.46-2.62 (2H, br dd, 6-H), 2.67 (1H, d,J=15.6 Hz, 1β-H), 2.92 (1H, d, J=15.6 Hz, 1α-H), 3.43 (3H, s, 3′-CH₃),3.68 (1H, dd, J_(16α,17α)=8.7 Hz, J_(16β,17α)=8.1 Hz, 17α-H) , 4.46-4.73(1H, m, 8′-CH_(a)H_(b)), 4.73-5.00 (1H, m, 8′-CH_(a)H_(b)), 6.39 (1H, s,4-H), 8.25 (1H, s, 5′-H)

Compound III-4(8′-n-Butyl-5′-deaza-17β-hydroxy-3′-methylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);yellow crystalline powder (0.79 g, 58%), mp 246° C. (decomp.); ¹H-NMR(300 MHz, CDCl₃) δ: 0.81 (3H, s, 18-CH₃), 0.99 (3H, s, 19-CH₃), 1.50(3H, t, J=7.2 Hz, 8′-CH₂CH₂CH₂CH₃) , 2.46-2.61 (2H, br dd, 6-H), 2.66(1H, d, J=15.6 Hz, 1β-H), 2.91 (1H, d, J=15.6 Hz, 1α-H), 3.45 (3H, s,3′-CH₃) , 3.69 (1H, dd, J_(16α,17α)=7.8 Hz, J_(16β,17α)=8.1 Hz, 17α-H),4.28-4.60 (1H, m, 8′-CH_(a)H_(b)), 4.60-4.92 (1H, m, 8′-CH_(a)H_(b)),6.35 (1H, s, 4-H), 8.25 (1H, s, 5′-H)

Compound II-5(8′-Benzyl-5′-deaza-17β-hydroxy-3′-methylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);yellow crystalline powder (0.63 g, 43%), mp 215° C.; ¹H-NMR (300 MHz,CDCl₃) δ: 0.78 (3H, s, 18-CH₃), 0.95 (3H, s, 19-CH₃), 2.37-2.45 (2H, brdd, 6-H), 2.64 (1H, d, J=15.5 Hz, 1β-H), 2.90 (1H, d, J=15.5 Hz, 1β-H),3.45 (3H, s, 3′-CH₃), 3.66 (1H, dd, J_(16α,17α)=8.4 Hz, J_(16β,17α)=8.5Hz, 17α-H), 5.59 (1H, br d, J=15.3 Hz, 8′-CH_(a)H_(b)), 6.26 (1H, br d,J=15.3 Hz, 8′-CH_(a)H_(b)), 6.28 (1H, s, 4-H), 7.07-7.19 (2H, m, Bn-mH),7.25-7.35 (3H, m, Bn-o, pH), 8.33 (1H, s, 5′-H)

Compound III-6(5′-Deaza-17β-hydroxy-3′-methyl-8′-phenylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);orange crystalline powder (0.54 g, 38%), mp 215° C. (decomp.); ¹H-NMR(300 MHz, CDCl₃) δ: 0.80 (3H, s, 18-CH₃), 1.01 (3H, s, 19-CH₃),2.20-2.43 (2H, br dd, 6-H), 2.76 (1H, d, J=15.9 Hz, 1β-H), 2.96 (1H, d,J=15.9 Hz, 1α-H), 3.40 (3H, s, 3′-CH₃), 3.67 (1H, dd, J_(16α,17α)=8.7Hz, J_(16β,17α)=8.9 Hz, 17α-H), 5.60 (1H, s, 4-H), 6.90-7.22 (2H, m,Ph-mH), 7.22-7.49 (3H, m, Ph-o, pH), 8.39 (1H, s, 5′-H)

Compound III-7(5′-Deaza-17β-hydroxy-3′-methyl-8′-(4-methylphenyl)androst-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);orange crystalline powder (0.58 g, 40%), mp 205° C. (decomp.); ¹H-NMR(300 MHz, CDCl₃) δ: 0.81 (3H, s, 18-CH₃), 1.02 (3H, s, 19-CH₃), 2.92(1H, d, J=15.9 Hz, 1β-H), 3.10 (1H, d, J=15.9 Hz, 1α-H), 2.46 (3H, s,8′-CH₃), 3.40 (3H, s, 3′-CH₃) , 3.67 (1H, dd, J_(16α,17α)=8.4 Hz,J_(16β,17α)=8.7 Hz, 17α-H), 5.56 (1H, s, 4-H), 6.88-7.20 (2H, m, Ar-mH),7.20-7.47 (2H, m, Ar-oH), 8.37 (1H, s, 5′-H)

Compound III-8(5′-Deaza-17β-hydroxy-8′-(4-methoxyphenyl)-3′-methylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);orange crystalline powder (0.69 g, 47%), mp 220° C. (decomp.); ¹H-NMR(300 MHz, CDCl₃) δ: 0.80 (3H, s, 18-CH₃), 1.02 (3H, s, 19-CH₃),2.18-2.41 (2H, br dd, 6-H), 2.70 (1H, d, J=15.6 Hz, 1β-H), 2.96 (1H, d,J=15.6 Hz, 1α-H), 3.40 (3H, s, 3′-CH₃), 3.67(1H, dd, J 8.7Hz,J_(16β,17α)=8.4 Hz, 17α-H), 3.89 (3H, s, OCH₃), 5.61 (1H, s, 4-H),7.02-7.24 (4H, m, Ar-m, oH), 8.36 (1H, s, 5′-H)

Compound III-9(5′-Deaza-17β-hydroxy-3′-methyl-8′-(3,4-methylenedioxyphenyl)-androst-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);orange crystalline powder (0.72 g, 47%), mp 214° C.; ¹H-NMR (300 MHz,CDCl₃) δ: 0.79 (3H, s, 18-CH₃), 1.01 (3H, s, 19-CH₃), 2.22-2.46 (2H, brdd, 6-H), 2.70 (1H, d, J=15.6 Hz, 1β-H), 2.96 (1H, d, J=15.6 Hz, 1α-H),3.40 (3H, s, 3′-CH₃) , 3.67 (1H, dd, J_(16α, 17α)=9.9 Hz,J_(16β,17α)=8.4 Hz, 17β-H), 5.67 (1H, s, 4-H), 6.08 (2H, s, OCH₂O) ,6.51-7.17 (1H, m, Ar-mH), 6.86-6.98 (2H, m, Ar-oH), 8.36 (1H, s, 5′-H)

Compound III-10(5′-Deaza-17β-hydroxy-8′-(4-hydroxyphenyl)-3′-methylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);orange crystalline powder (0.47 g, 32%), mp 280° C. (decomp.); ¹H-NMR(300 MHz, CDCl₃) δ: 0.79 (3H, s, 18-CH₃), 1.00 (3H, s, 19-CH₃),2.61-2.78 (2H, br dd, 6-H), 2.71 (1H, d, J=15.6 Hz, 1β-H), 2.98 (1H, d,J=15.6 Hz, 1α-H), 3.45 (3H, s, 3′-CH₃) , 3.67 (1H, dd, J_(16α,17α)=7.6Hz, J_(16β,17α)=8.4 Hz, 17α-H) , 5.68 (1H, s, 4-H), 6.73-6.98 (4H, m,Ar-m, oH), 8.40 (1H, s, 5′-H)

Compound III-11(5′-Deaza-8′-(4-fluorophenyl)-17β-hydroxy-3′-methylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);orange crystalline powder (0.69 g, 47%), mp 270° C. (decomp.); ¹H-NMR(300 MHz, CDCl₃) δ: 0.80 (3H, s, 18-CH₃), 1.00 (3H, s, 19-CH₃) , 2.68(2H, dd, J=15.0, 6-H), 2.93 (1H, d, J=15.0 Hz, 1β-H), 3.11 (1H, d,J=15.0 Hz, 1α-H), 3.70 (3H, s, 3′-CH₃), 3.56-3.80 (1H,br dd, 17α-H),6.54 (1H, s, 4-H), 7.12-7.66 (4H, m, Ar-m, oH), 8.50 (1H, s, 5′-H)

Compound III-12(8′-(4-Chlorophenyl)-5′-deaza-17β-hydroxy-3′-methylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);yellow crystalline powder (0.74 g, 49%), mp 270° C. (decomp.); ¹H-NMR(300 MHz, CDCl₃) δ: 0.80 (3H, s, 18-CH₃), 1.03 (3H, s, 19-CH₃),2.19-2.42 (2H, br dd, 6-H), 2.70 (1H, d, J=15.6 Hz, 1β-H), 3.96 (1H, d,J=15.6 Hz, 1α-H), 3.40 (3H, s, 3′-CH₃), 3.67 (1H, dd, J^(16α,17α)=8.4Hz, J_(16β,17α)=8.4 Hz, 17α-H), 5.53 (1H, s, 4-H), 7.06-7.21 (2H, m,Ar-mH), 7.50-7.60 (2H, m, Ar-oH), 8.37 (1H, s, 5′-H)

Compound III-13(8′-(4-Bromophenyl)-5′-deaza-17β-hydroxy-3′-methylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);orange crystalline powder (0.72 g, 44%), mp 250° C. (decomp.); ¹H-NMR(300 MHz, CDCl₃) δ: 0.80 (3H, s, 18-CH₃), 1.03 (3H, s, 19-CH₃),2.25-2.42 (2H, br dd, 6-H), 2.70 (1H, d, J=15.3 Hz, 1β-H), 3.96 (1H, d,J=15.3 Hz, 1α-H), 3.39(3H, s, 3′-CH₃), 3.67 (1H, dd, J_(16α,17α)=8.7 Hz,J_(16β,17α)=8.1 Hz, 17α-H) , 5.53 (1H, s, 4-H), 6.99-7.15 (2H, m,Ar-mH), 7.65-7.75 (2H, m, Ar-oH), 8.38 (1H, s, 5′-H)

PRODUCTION EXAMPLE 2

(General synthesis of8′-substituted-5′-deaza-17β-hydroxy-3′-phenylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione derivatives (from III-14 to III-23))

To diphenyl ether (1 ml) were added p-toluenesulfonic acid (60 mg, 0.32mmol), 6-(monosubstituted amino)-3-phenyluracil (1) (0.66 g, 2.84 mmol)and 2-hydroxymethylene testosterone (6) (1.0 g, 3.16 mmol) and theresulting mixture was stirred at 155° C. for 45 minutes in a nitrogenatmosphere. After the reaction, the reaction product was separated andpurified by column chromatography (Fuji Silysia from 230 to 400 mesh;eluent: ethyl acetate:ethanol=10:1 or ethyl acetate alone) to obtaincrystalline powder. Further, recrystallization can be carried out usinga mixed solution of ethyl acetate and n-hexane.

Compound III-14(5′-Deaza-8′-ethyl-17β-hydroxy-3′-phenylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione); yellow crystalline powder (0.79g, 54%), mp 240° C.(decomp.); ¹H-NMR (300 MHz, CDCl₃) δ: 0.81 (3H, s, 18-CH₃), 1.01 (3H, s,19-CH₃), 1.45 (3H, t, J=7.5 Hz, 8′-CH₂CH₃), 2.52-2.61 (2H, br dd, 6-H),2.67 (1H, d, J=15.6 Hz, 1β-H), 2.93 (1H, d, J=15.6 Hz, 1α-H), 3.68 (1H,dd, J_(16α,17α)=7.5 Hz, J_(16β,17α)=8.1 Hz, 17α-H) , 4.46-4.76(1H, m,8′-CH_(a)H_(b)), 4.76-5.05 (1H, m, 8′-CH_(a)H_(b)), 6.41 (1H, s, 4-H),7.21-7.45 (2H, m, Ph-mH), 7.45-7.52 (3H, m, Ph-o, pH), 8.27 (1H, s,5′-H)

Compound III-15(8′-n-Butyl-5′-deaza-17β-hydroxy-3′-phenylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione); yellow crystalline powder(0.77g, 50%), mp 200° C.; ¹H-NMR (300 MHz, CDCl₃) δ: 0.78 (3H, s,18-CH₃), 0.99 (3H, s, 19-CH₃), 1.18 (3H, t, J=7.2 Hz, 8′-CH₂CH₂CH₂CH₃),2.45-2.62 (2H, br dd, 6-H), 2.65 (1H, d, J=15.6 Hz, 1β-H), 2.91(1H, d,J=15.6 Hz, 1α-H), 3.63 (1H, dd, J_(16α,17α)=7.1 Hz, J_(16β,17α)=7.1 Hz,17α-H), 4.35-4.64 (1H, m, 8′-CH_(a)H_(b)), 4.64-4.94 (1H, m,8′-CH_(a)H_(b)), 6.39 (1H, s, 4-H), 7.21-7.52 (5H, m, Ph-o, m, pH), 8.27(1H, s, 5′-H)

Compound III-16(8′-Benzyl-5′-deaza-17β-hydroxy-3′-phenylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);yellow crystalline powder (0.72 g, 44%), mp 224° C.; ¹H-NMR (300 MHz,CDCl₃) δ: 0.81 (3H, s, 18-CH₃), 0.92 (3H, s, 19-CH₃), 2.30 (2H, dd,J=10.5 Hz, 6-H), 2.63 (1H, d, J=15.6 Hz, 1β-H), 2.87 (1H, d, J=15.6 Hz,1α-H), 3.67 (1H, dd, J_(16α,17α)=8.4 Hz, J_(16β,17α)=7.8 Hz, 17α-H),5.37-5.61 (1H, br, 8′-CH_(a)H_(b)), 6.41-6.64 (1H, br, 8′-CH_(a)H_(b)),7.02 (1H, s, 4-H), 7.24-7.56 (10H, m, Bn-o, m, pH and Ph-o, m, pH), 8.33(1H, s, 5′-H)

Compound III-17(5′-Deaza-17β-hydroxy-3′,8′-diphenylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);orange crystalline powder (0.57 g, 36%), mp 257° C.; ¹H-NMR (300 MHz,CDCl₃) δ: 0.79 (3H, s, 18-CH₃), 1.04 (3H, s, 19-CH₃), 2.71 (1H, d,J=15.6 Hz, 1β-H), 2.98 (1H, d, J=15.6 Hz, 1α-H), 3.65 (1H, dd,J_(16α,17α)=8.4Hz, J_(16β,17α)=8.7 Hz, 17α-H), 5.57 (1H, s, 4-H),7.16-7.66(10H, m, 3′-Ph-o, m, pH and 8′-Ph-o, m, pH), 8.40 (1H, s, 5′-H)

Compound III-18(5′-Deaza-17β-hydroxy-8′-(4-methylphenyl)-3′-phenylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);orange crystalline powder (0.90 g, 55%), mp 246° C. (decomp.); ¹H-NMR(300 MHz, CDCl₃) δ: 0.79 (3H, s, 18-CH₃), 0.88 (3H, s, 19-CH₃), 2.47(3H, s, 8′-CH₃), 2.58-2.74 (2H, br dd, 6-H), 2.92 (1H, d, J=15.0 Hz,1β-H), 3.16 (1H, d, J=15.0 Hz, 1α-H), 3.68 (1H, dd, J_(16α,17α)=8.7 Hz,J_(16β,17α)=7.8 Hz, 17α-H) , 6.61 (1H, s, 4-H), 6.89-7.49 (7H, m,3′-Ph-m, pH and 8′-Ar-o, mH), 8.40-8.56 (2H, m, 3′-Ph-oH), 8.49 (1H, s,5′-H)

Compound III-19(5′-Deaza-17β-hydroxy-8′-(3,4-dimethylphenyl)-3′-phenylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);orange crystalline powder (0.90 g, 54%), mp 260° C. (decomp.); ¹H-NMR(300 MHz, CDCl₃) δ: 0.77 (3H, s, 18-CH₃), 0.80 (3H, s, 19-CH₃), 2.33(3H, s, 8′-Ar-CH₃), 2.35 (3H, s, 8′-Ar-CH₃), 2.71(1H, d, J=15.0 Hz,1β-H), 2.97 (1H, d, J=15.0 Hz, 1α-H), 3.67 (1H, dd, J_(16α,17α)=7.5 Hz,J_(16β,17α)=8.4 Hz, 17α-H) , 5.60 (1H, s, 4-H), 6.88-7.50 (8H, m,3′-Ph-o, m, pH and 8′-Ar-o, mH), 8.38 (1H, s, 5′-H)

Compound III-20(5′-Deaza-17β-hydroxy-8′-(4-methoxyphenyl)-3′-phenylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);orange crystalline powder(0.96 g, 57%), mp 250° C. (decomp.); ¹H-NMR(300 MHz, CDCl₃) δ: 0.78 (3H, s, 18-CH₃), 0.81 (3H, s, 19-CH₃),2.23-2.38 (2H, br dd, 6-H), 2.71 (1H, d, J=15.6 Hz, 1β-H), 2.93 (1H, d,J=15.6 Hz, 1α-H), 3.62 (1H, dd, J_(16α,17α)=6.9 Hz, J_(16β,17α)=6.6 Hz,17α-H), 3.87 (3H, s, OCH₃), 6.19 (1H, s, 4-H), 7.00-7.13 (7H, m,3′-Ph-m, pH and 8′-Ar-o, mH), 7.17-7.50 (2H, m, 3′-Ph-oH), 8.38 (1H, s,5′-H)

Compound III-21(5′-Deaza-8′-(4-fluorophenyl)-17β-hydroxy-3′-phenylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);orange crystalline powder(0.61 g, 37%), mp 260° C. (decomp.); ¹H-NMR(300 MHz, CDCl₃) δ: 0.79 (3H, s, 18-CH₃), 1.04 (3H, s, 19-CH₃), 2.70(1H, d, J=15.9 Hz, 1β-H), 2.97 (1H, d, J=15.9 Hz, 1α-H), 3.66 (1H, dd,J_(16α,17α)=8.1 Hz, J_(16β,17α)=8.7 Hz, 17α-H), 5.54 (1H, s,4-H),7.18-7.54 (10H, m, 3′-Ph-o, m, pH and 8′-Ph-o, m, pH), 8.39 (1H, s,5′-H)

Compound III-22(8′-(4-Chlorophenyl)-5′-deaza-17β-hydroxy-3′-phenylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);orange crystalline powder (0.56 g, 33%), mp 241° C. (decomp.); ¹H-NMR(300 MHz, CDCl₃) δ: 0.77 (3H, s, 18-CH₃), 0.85 (3H, s, 19-CH₃),2.29-2.44 (2H, br dd, 6-H), 2.79 (1H, d, J=16.2 Hz, 1β-H), 3.07 (1H, d,J=16.2 Hz, 1α-H), 3.67 (1H, dd, J_(16α,17α)=7.8 Hz, J_(16β,17α)=8.4 Hz,17α-H), 6.46 (1H, s, 4-H), 7.17-7.59 (10H, m, 3′-Ph-o, m, pH and8′-Ph-o, m, pH), 8.47 (1H, s, 5′-H)

Compound III-23(8′-(4-Bromophenyl)-5′-deaza-17β-hydroxy-3′-phenylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-dione);orange crystalline powder (0.93, 51, mp 300° C. (decomp.); ¹H-NMR (300MHz, CDCl₃) δ: 0.81 (3H, s, 18-CH₃), 0.83(3H, s, 19-CH₃), 2.69 (1H, d,J=15.0 Hz, 1β-H), 3.12 (1H, d, J=15.0 Hz, 1α-H), 3.07 (1H, dd,J_(16α,17α)=7.9 Hz, J_(16β,17α)=8.7 Hz, 17α-H), 6.56 (1H, s, 4-H),6.69-7.52 (7H, m, 3′-Ph-m, pH and 8′-Ar-o, mH), 7.67-7.79 (2H, m,3′-Ph-oH), 8.48 (1H, s, 5′-H)

Instrumental analysis values of the above novel compounds (from III-3 toIII-23) are shown in Table 5 and Table 6.

TABLE 5 Physical data of 8′-substituted5′-deaza-17β-hydroxy-3′-methylandrosi-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-diones (III-3−III-13)        Compd. No.         R         Yield (%)

 Formula         Mp (° C.)

III-3 Et 60 260 c

(decomp.) III-4 Bu 58 246 72.92 8.23 8.80

(72.88 7.84 9.17) III-5 Bn 43 215 71.35 7.48 7.80

(71.55 7.30 7.83) III-6 Ph 38 215 70.72 7.31 7.98

(decomp.) (70.32 7.13 8.08) III-7 4-Me-C₆H₄ 40 205 74.46 7.32 8.14

(decomp.) (74.25 6.93 8.17) III-8 4-MeO-C₆H₄ 46 220 70.44 7.20 7.70

(decomp.) (70.06 7.15 8.05) III-9 3,4-OCH₂O-C₆H₄ 47 214 68.24 6.69 7.46

(decomp.) (68.26 6.49 7.24) III-10 4-HO-C₆H₄ 32 280 71.24 6.94 8.04

(decomp.) (71.04 6.74 8.23) III-11 4-F-C₆H₄ 47 270 68.61 6.87 7.74

(decomp.) (68.54 6.48 7.79) III-12 4-Cl-C₆H₄ 49 270 68.81 6.52 7.77

(decomp.) (68.48 6.20 7.64) III-13 4-Br-C₆H₄ 44 250 63.59 6.02 7.18

(decomp.) (63.88 6.06 7.18) ^(a)All compounds were obtained as yellowpowder. ^(b)All compounds were recrystallized from EtOAc-n-hexane.^(c)Mass Spectrum: MH

 = 450 (Matrix:Gly)

indicates data missing or illegible when filed

TABLE 6 Physical data of 8′-substituted5′-deaza-17β-hydroxy-3′-phenylandrost-2,4-dieno[2,3-g]pteridine-2′,4′(3′H,8′H)-diones (III-14−III-23)        Compd. No.         R         Yield (%)

 Formula         Mp (° C.)

III-14 Et 54 240 (decomp.) 73.82 7.36 8.07

(73.48 7.64 7.94) III-15 Bu 50 200 73.46 7.76 7.56

(73.25 7.66 7.77) III-16 Bn 44 224 75.10 6.98 7.10

(75.38 7.06 7.45) III-17 Ph 36 257 75.63 6.76 7.35

(75.55 6.58 7.69) III-18 4-Me-C₆H₄ 55 246 73.97 7.05 6.99

(73.78 6.66 7.33) III-19 3,4-Me₂-C₆H₄ 54 260 (decomp.) 75.72 7.13 6.97

(75.49 6.75 6.83) III-20 4-MeO-C₆H₄ 57 250 (decomp.) 73.85 6.76 6.98

(73.54 6.75 7.07) III-21 4-F-C₆H₄ 37 260 (decomp.) 73.70 6.36 7.16

(73.81 6.72 6.97) III-22 4-Cl-C₆H₄ 33 241 (decomp.) 70.89 6.24 6.89

(71.06 6.48 6.50) III-23 4-Br-C₆H₄ 51 300 (decomp.) c

^(a)All compounds were obtained as yellow powder. ^(b)All compounds wererecrystallized from EtOAc-n-hexane. ^(c)Mass Spectrum: MH

 = 640, MH

 +2 = 642 (Matrix:Gly)

indicates data missing or illegible when filed

Pyridodipyrimidine compounds (IV) (from IV-1 to IV-26) (9) representedby the formula IV can be synthesized by a production method described inthe known document (22) (Production method D). Described specifically,p-toluenesulfonic acid, 3-substituted-6-monosubstituted aminouracil (1),and 2-hydroxymethylenecholest-4-en-3-one (8) are added to diphenyl etherand the resulting mixture is heated and stirred at 180° C. for 45minutes in a nitrogen atmosphere. After the reaction, the reactionproduct is separated and purified by column chromatography (FujiSilysia, from 230 to 400 mesh; eluent: ethyl acetate) to obtaincrystalline powder.

Next, direct or indirect contribution of the addition of each ofCompounds No. I-50 (Specimen 1), I-54 (Specimen 2), III-4 (Specimen 3),and I-75 (Specimen 4) to intracellular ATP production was verified by anexperiment, so that it will next be described in Examples.

EXAMPLE 1

After the addition of Specimen 1 to cultured cells of a human-derivedneuroblast (Neuroblastoma) SH-SYSY strain, fluorescence measurement ofan intracellular/extracellular ATP concentration was performed. Theresults are shown in FIG. 1. In the cells, an increase in ATP production(increase in fluorescence intensity) is observed for 5 hours immediatelyafter the addition of Specimen 1 (1 μM) and the effect disappears after12 hours. It is to be noted that no morphological change of the culturedcells by the treatment with the specimen is observed.

Also outside the cells, an increase in ATP production is observed for 5hours immediately after the addition of Specimen 1 (1 μM) and an ATPconcentration increases, which is presumed to occur due to the outflowof ATP, which has shown an increase in the cells, therefrom.

EXAMPLE 2

Specimen 1 was added to astrocytes, one of glial cells of the centralnerve system, after culturing and the fluorescence measurement resultsare shown in FIG. 2A and FIG. 2B.

The ATP amount in the glial cells significantly increased 12 hours and24 hours after the administration of 1 μM of Specimen 1 and anextracellular amount also shows a similar tendency.

EXAMPLE 3

To cultured human glioma US251 cells (Sigl-R transfected cells) wereadded Specimens 1 and 2, respectively and intercomparison was performedbetween intracellular ATP concentration after addition and that withoutaddition (Cont.). As a result, it was verified as shown in FIG. 3 thatSpecimen 1 and Specimen 2 significantly increased the ATP productionamount of human glioma U251 cells. Specimen 2 having higherlipophilicity is more effective than Specimen 1. It is to be noted thatfluorescence measurement was performed 6 hours after the administrationof the specimens.

In Examples 1 to 3, an experiment on brain neurons (neurons) wasperformed. The brain neurons (neurons) require oxygen (O₂) and glucose.These two substances are first taken into glias (astrocytes) from theblood and then, transmitted from the glias to neurons. The specimen alsoshows similar transmission. An increase in ATP amount in the gliascaused by the addition of the specimen therefore shows direct promotionof the activity of the glias and at the same time, suggests indirectactivation of the neurons.

EXAMPLE 4

Hippocampal neurons were collected from a juvenile (0 day after birth)ICR mouse brain and neurons were cultured on a culture dish.

Specimen 1 was added once to the cells in the initial stage of growth(on culture day 1) and after three days (on culture day 4), the numberof neuron axons, dendrites , and growing synapses of the hippocampalneurons (neurons) was quantified. Specimen 1 (0.1 μM, 0.3 μM, 1.0 μM)was added once on culture day 1. Axon and Dendrite were immunostainedwith a tau antibody and a MAP2 antibody, respectively, three days afterthe addition (on culture day 4) and morphology thereof was observed.

FIG. 4 shows immunostained images of the hippocampal neuron axon. Onculture day 4, the axon was immunostained with a tau antibody. In theseimages, a left one shows Control (control group) and a right one shows aspecimen-added neuron obtained by adding Specimen 1 (1 μM) once onculture day 1.

FIG. 5 shows the the number of branches of hippocampal neuron axons andthe number of branches of the axons stained with a tau antibody wasquantified on culture day 4. An experiment was performed by drawingconcentric circles (not shown) at an interval of 10 μm with a soma as acenter and the number of axons crossing the circles was measured.

FIGS. 4 and 5 show that Specimen 1 concentration-dependently increasesthe elongation/growth of neuron axons and the number of branches. Therespective specimens having a concentration of 0.3 μM and 1.0 μMexhibited almost similar maximum drug efficacy.

FIG. 6 shows an immunostained image of a hippocampal neuron dendrite.The dendrite was immunostained with a MAP2 antibody on culture day 4. Aleft image shows Control (control group) and a right image shows neuronsto which Specimen 1 (1 μM) was added once on culture Day 1.

FIG. 7 shows the number of branches of a hippocampal neuron dendrite.The dendrite was immunostained with a MAP2 antibody on culture day 4 andthe number of branches of the dendrite was quantified. An experiment wasperformed by drawing concentric circles (not shown) at an interval of 10pm with a soma as a center and the number of dendrites crossing thecircles was counted.

FIGS. 6 and 7 show that Specimen 1 concentration-dependently extendedthe dendrite and increased the number of branches thereof. Therespective specimens having concentrations of 0.3 μM and 1.0 μM showedalmost similar maximum drug efficacy.

Then, excitatory synapses on culture 14 day were immunostained with aVGLUT1 antibody and the number of synapses was quantified.

FIG. 8 shows an immunostained image of a synapse in an autapsepreparation. The left image shows Control (control group) and the rightimage shows neurons obtained by adding Specimen 1 (1 μM) only once onculture day 1 and then immunostaining an excitatory synapse with aVGLUT1 antibody on culture day 14.

FIG. 9 shows an increase in excitatory synapse projected to hippocampalneurons. The brain has two synapses, that is, glutamatergic andGABAergic synapses which speedily transmit excitation and inhibition ofneurons. FIG. 9 shows the quantified number of excitatory synapsesimmunostained with a VGLUT1 antibody on culture day 14.

FIGS. 8 and 9 show that addition of Specimen 1 in an amount of 0.3 μMsignificantly increased the number of excitatory synapses to themaximum.

EXAMPLE 5

As in Example 4, hippocampal neurons were collected from a juvenile (0day after birth) ICR mouse brain and the neurons were cultured on aculture dish.

Specimen 1 was administered once to the mature cultured hippocampalcells (on culture day 11) and three days later (on culture day 14), theneuron axons and dendrites of the hippocampal neurons (neurons) wereobserved. The axons on culture day 14 showed marked elongation andmarked crossings therebetween and the number of the axons cannot bequantified, so that only the morphology of the dendrites was observed.

FIG. 10 includes immunostained images of the hippocampal neurondendrite. A left one is an image of Control (control group) and a rightone is an image on culture day 14 obtained by administering Specimen 1and immunostaining with a MAP2 antibody on culture day 14.

FIG. 11 shows the number of branches of a dendrite immunostained with aMAP2 antibody on culture day 14, that is, in the latter growth stage andquantified at a specimen concentration of from 0.1 to 1 μM. Concentriccircles were drawn at an interval of 10 μm with a soma as a center andthe number of dendrites crossing the circles was measured.

Compared with the number of dendrites of the immature cultured cells onday 4 (FIG. 7), the number of dendrites growing from the soma increases,depending on the number of culturing days (FIG. 11). Although the numberof dendrites tends to increase even in the mature cultured cell atconcentrations of the specimen added (0.3 μM and 1 μM), an increase inthe number of branches was not so more marked than that caused by thedrug efficacy observed in the cells on culture day 4.

The experimental results in Examples 4 and 5 have indirectly proved thatdue to the addition of the specimen, increase or promotion of thegrowth/branching/the number of synapses of immature/mature culturedneurons of rats is promoted by increased ATP production in neurons.

EXAMPLE 6

One of adult C57BL/6N mice (from 10 to 17 weeks old, weight: from 28 to31 g) was intraperitoneally administered with physiological saline(Control) and the other one with Specimen 3 (10 μg/kg). After 22 hours,a thin brain slice preparation of each mouse including the cortex andthe hippocampus was made. Depolarization of a cellular membrane wascaused by the addition of 80 mM KCl (80K) to an extracellular fluid andan increase in mitochondrial Ca²⁺ concentration resulting therefrom wasmeasured by a fluorescent method with Rhod-2 (Note 1). Positiveparticipation of Specimen 3 to the ATP activity in mitochondria wasindirectly verified (Note 3) by using, as an index, how the Ca²⁺concentration to be increased by 80K (Note 2) is suppressed by Specimen3 in the brain slice.

(Note 1) Rhod-2: Ca²⁺ fluorescent dye selectively incorporated inmitochondria

(Note 2) The depolarization of the neuronal membrane by 80K occurs dueto both the flow of Ca²⁺ from the outside to the inside of the cell andthe release of Ca²⁺ from an intracellular Ca store. The free Ca²⁺ whichhas increased in the cell easily enters the mitochondria, anintracellular independent organ. This results in an increase in themitochondrial Ca²⁺ concentration.

(Note 3) The free Ca²⁺ which has increased in the neuronal cytoplasm dueto 80K stimulation-induced depolarization speedily and immediatelytransmits into the mitochondria from the cytoplasm. A decrease in themitochondrial free Ca²⁺ concentration caused by the specimen occursbecause the free Ca²⁺ is scooped out of the cell by an outward Ca pumppresent in the neuron cytoplasm membrane or adsorption and fixing to aportion inside the mitochondria. Energy for it is supplied from ATPproduced in the mitochondria. Based on the results thus obtained,intraperitoneally injected Specimen 3 passes from the blood to glia(astrocyte) cells and is then, incorporated in the mitochondria in thecerebral cortex or hippocampal neuron, by which ATP production in themitochondria is activated and neurons are activated. The Ca pump on theneuron cytoplasm gets ATP, an energy source, from the mitochondria,while discharges extra intracellular free Ca²⁺ out of the cell andindirectly protects neurons.

FIG. 12 shows the measurement of a K⁺ depolarization-induced Ca²⁺concentration increase in mitochondria present in the cerebral cortexneurons (neurons) and an inhibitory effect of Specimen 3 thereon.

a shows a control reaction when a brain slice preparation obtained froma normal mouse is treated with a 80 mM KCl extracellular fluid(application for 5 minutes, followed by washing for 5 minutes) threesuccessive times. Fluorescence measurement results of a mitochondrialCa²⁺ increase are plotted along the ordinate.

b shows variations of a mitochondrial Ca²⁺ concentration when a 80Kextracellular fluid is given to a brain slice preparation excised andobtained from a rat 22 hours after it was intraperitoneally (i.p.)injected with 10 μg/kg of Specimen 3. Compared with a, suppression ofthe Ca²⁺ concentration increase and a faster recovery from the Ca²⁺concentration increased by 80K depolarization can be observed.

c shows a comparison between Control and Specimen 3 in the effect on themitochondrial Ca²⁺ concentration in the cerebral cortex neuron. Themaximum efficacy for suppressing the Ca²⁺ concentration at 10 μm/kg isobserved (the same results as those at an injection of 10 mg/kg, 1000times the amount) (n=3 average: data obtained from three slices obtainedfrom the same individual).

FIG. 13 shows the measurement of a K⁺ depolarization-inducedmitochondrial Ca²⁺ concentration increase in hippocampal neurons(neurons).

a shows a control reaction when a 80 mM KCl extracellular fluid(application for 5 minutes, followed by washing for 5 minutes) is giventhree successive times.

b shows variations of a mitochondrial Ca²⁺ concentration when a 80Kextracellular fluid is given to a brain slice preparation made 22 hoursafter intraperitoneal injection of 10 μg/kg of Specimen 3.

c shows a comparison among Control and 10 μg/kg and 10 mg/kg i.p. ofSpecimen 3 in the inhibitory effect on a mitochondrial Ca²⁺concentration increase in hippocampal neurons. The maximum effect isattained at 10 μg/kg (n=3, average: data from three slices obtained fromthe same individual).

Based on the experimental results in Example 6, the 10 μg/kg i.p.pretreatment in vivo with Specimen 3 decreases a 80Kdepolarization-induced increase in mitochondrial Ca²⁺ concentration inthe mouse cortex and hippocampal neurons (neurons) markedly and at themaximum. These results suggest that Specimen 3 relieves the damage ofneurons which has occurred at the time of cerebral ischemia or the like.

This suggests that against a Ca load due to a mitochondrial free Ca²⁺increase that damages adult mouse brain neurons, an ATP increase causedby Specimen 3 activates a Ca pump on the cellular membrane, decreasesthe mitochondrial free Ca²⁺ amount, and thereby contributes toprevention of neuronal apoptosis (indirect proof of ATP increase).

EXAMPLE 7

An experiment was made for confirming the effect of the addition of aspecimen to cerebral ischemia model adult rats having deterioratedexercise performance.

Intracerebral hemorrhage model rats were prepared by making a small holeinto the skull of adult Wistar rats (weight: from 200 to 230 g) underinhalation anesthesia, administering 1.2 μl of a physiological saline tothe right brain striatum in Control group and a physiological salinecomprising 0.24U collagenase (type IV) to that of a hemorrhage group.

FIG. 14 shows the respective brain cross-sections of the intracerebralhemorrhage model rats.

In order to confirm the brain cell protective action of Specimen 4 forthe intracerebral hemorrhage model rats, 100 μg/kg of Specimen 4 wasinjected into a spot (white lozenge in the drawing) one hour afteradministration of collagenase, and exercise footprints of the rat werequantitatively measured.

For the measurement of the amount of exercise, the moving image of therats was photographed for 5 minutes. The free movement (exercisedistance and exercise speed) during the latter 3 minutes of thephotographing time was analyzed.

FIG. 15 shows the measurement of exercise footprints of the rats, whileFIG. 16 shows the measurement of daily changes of the exercise distanceand exercise speed. Specimen 4 ameliorates an ischemic decrease inexercise distance and exercise speed that occurs due to the cerebralischemia of the rat (FIGS. 15 and 16). This effect can be observed bothin hemorrhage day 1 and one week after hemorrhage.

The experimental results suggest that Specimen 4 contributes toprevention of ischemic damage of adult rat brain cells (indirect proofof ATP increase).

Next, tested was whether the addition of the following compounds as aspecimen: Compounds No. 11-13 (Specimen 5), II-31 (Specimen 6), III-2(Specimen 7), III-14 (Specimen 14), IV-10 (Specimen 9), IN-15 (Specimen10), and IV-23 (Specimen 11) significantly increased the number ofbranches of a hippocampus neuron dendrite or not.

Hippocampal neurons were collected from the juvenile (Day 0 after birth)ICR mouse brain and cultured on a culture dish.

Each specimen was added once to the cells in the initial growth stage(on culture day 1); the resulting cells were immunostained with a MAP2antibody three days later (on culture day 4), and the number of branchesof the dendrite was quantified.

The measurement was performed by drawing 20 μm to 100 μm concentriccircles (not shown) at an interval of 10 μm with a soma as a center andcounting the number of dendrites crossing the circles.

A line graph of the number of dendrite crossings counted for eachdistance from the soma was drawn and a Student's t-test, paired (t-test,two tailed distribution) was carried out using an area under the linegraph (Area under the Curve: AUC).

When the number of cases of a specimen was sufficient, the test wasperformed while eliminating the maximum and minimum values. Theconcentrations of the specimens to be added were each 0.3 μM.

Whether the dendrite showed significant elongation or not was judged bya p-value calculated from a t-value as follows: when p<0.05, thedendrite showed significant elongation (effect ++); when 0.05<p<0.2, theelongation was at distal or proximal site (effect +); and when p>0.2,there was no significant difference (effect: ±).

FIGS. 22 to 28 show the results of adding Specimens 5 to 10,respectively and carrying out a quantitative test of the number ofbranches of the hippocampal neuron dendrite.

In these drawings, a shows the number of dendrite crossings relative toa distance from soma and b shows the test results obtained by comparisonin AUC.

A solid line shows a specimen addition case and a dotted line shows acase where a specimen is not added (control). It is to be noted that nis the number of preparations.

As is apparent from the graphs, a statistically significant differenceis recognized (p<0.05) only from Specimen 7 (III-2) and Specimen II(IV-23) and an effect on dendrite elongation is recognized also from theother specimens, that is, 5 (II-13), 6 (II-31), 8 (III-2), 9 (IV-10), 10(IV-15), and 11 (IV-23).

Next, Specimen 1 (TND1128) was selected and a comparison experiment ineffect between it and β-NMN was made. In the experiment, compared andevaluated is their inhibitory effect on marked variations of cytoplasmicand mitochondrial Ca²⁺ concentrations caused by exposure of mouse brainslice preparations to severe depolarization stimulation. A preliminaryexperiment by the present inventors and the like provided the findingthat SIRT1 showed a significant increase in a nematode treated for 24hours with Specimen 1 (TND1128), so that comparison was made to knowwhether a similar effect is achieved by β-NMN 24 hours afteradministration thereof.

Experimental Method

Formation of brain slice preparation and measurement of calciumconcentration: Used was a whole brain half-cut preparation obtained bycutting a whole brain slice (300 μm) of a mouse (C57B/6NL), pretreatedwith Specimen 1 for 24 hours by subcutaneous administration, at a medianline. The preparation was stored at room temperature in an artificialcerebrospinal fluid (ACSF) aerated with 95% O₂ and 5% CO₂ and was doublestained with Xrhod-1/AM (Kd=700 μM) which would be incorporatedselectively in mitochondria and fura-4F/AM (Kd=770 μM) which wouldremain in the cytoplasm. The resulting preparation was placed in achamber provided on a stage of an inverted fluorescence microscope(Olympus IX71), covered with a piece of black cotton, fixed with aplatinum ring, and refluxed (70 ml/hr) in ACSF aerated with 95% O₂ and5% CO₂. A fluorescent image of the preparation was acquired using a 4×objective lens and respective images of the hippocampal ventral part andcerebral cortex temporal region were obtained in one screen. For themeasurement of a mitochondrial calcium concentration, a change inintensity of red fluorescence (>600 nm) caused by excitation light at580 nm was determined and for the measurement of a cytoplasmic calciumconcentration, a ratio of fluorescence (>500 nm) between excitation at360 nm and that at 380 nm was determined. A hippocampal CA1 site and acerebral cortex were used as two regions of interest (ROI).

Drug Preparation Method and Administration Method

In water was dissolved β-NMN to give a 10 mg/kg, 30 mg/kg, or 100 mg/kgsolution; Specimen 1 was dissolved in an alcohol to give a solution of 1mg/ml and then the resulting solution was diluted with water to give a0.01 mg/kg, 0.1 mg/kg or 1.0 mg/kg solution; and they weresubcutaneously administered to mice 24 hours before the formation of thepreparation (0.1 ml/10 g).

Efficacy Evaluation Method

In the present test, an operation of exposing the brain preparationsformed from mice administered with two drugs having respectiveconcentrations to isotonic 80 mM KCl-ACSF (artificial cerebrospinalfluid) for 5 minutes and then returning them in normal ACSF for 5minutes was performed three successive times to give a severe calciumload to the preparations. Variations in cytoplasmic and mitochondrialcalcium concentrations at that time were measured.

Experimental Results

Effects of β-NMN and Specimen 1 (TND1128) on variations of mitochondrialcalcium concentration

FIG. 29 shows variations of a mitochondrial Ca²⁺ concentration when thepreparations formed from mice pretreated for 24 hours by subcutaneousadministration with β-NMN (A: 0 (control) (n=6), B: 10 (n=5), C: 30(n=5), and D: 100 mg/kg (n=5)) were exposed to 80K ACSF three times.Similarly, FIG. 30 shows variations of a mitochondrial calciumconcentration when the brain preparations of mice administered withSpecimen 1 (TND1128) (A: 0 (control) (n=6), B: 0.01 (n=5), C: 0.1 (n=5),and D: 1.0 mg/kg (n=5) s.c.) were exposed to 80K ACSF. The response ofthe preparations obtained from individuals treated with both drugs atrespective doses is normalized (normalized) with fluorescence intensityat the first 80K administration time as a standard and a time average ofthe cytoplasmic or mitochondrial Ca²⁺ concentration and a standarddeviation thereof are shown. It is obvious from these FIGS. 29 and 30that these drugs dose-dependently suppress a mitochondrial Ca²⁺ increasewithin a range of their doses.

FIG. 31 and FIG. 32 show the quantification results of the effects ofβ-NMN and Specimen 1 (TND1128) at each concentration. In FIG. 31 andFIG. 32, A shows the results of calculating a mitochondrial calciumconcentration, as AUC (Area under the curve), for 35 minutes from 5minutes after starting of the experiment by administration to threetimes exposure to 80K-ACSF and 5-minute washing (refer to FIG. 37). Band C show the drug effects on 5-minute 80K administration and 5-minutesubsequent washing (1st: from 5 minutes to 15 minutes, 2nd: from 15minutes to 25 minutes, and 3rd: from 25 minutes to 35 minutes) in thecerebral cortex (CTX) and the hippocampus (CA1), respectively (refer toFIG. 37). A significant difference test was performed using Tukey'smethod. Within the dose range thus studied, a dose response relationshipof β-NMN and TND1128 was observed.

Effects of β-NMN and Specimen 1 (TND1128) on Variations in CytoplasmicCalcium Concentration

FIG. 33 and FIG. 34 show the effects of β-NMN and Specimen 1 (TND1128)on cytoplasmic calcium variations at the time of administration of 80KACSF three successive times. In the Control response shown in FIG. 33and FIG. 34, the response recovery is small at each 80K-ACSFadministration time and the response of a calcium indicator seems toreach its peak. An intracellular calcium concentration indicator fura-4Fused in the test has a Ca²⁺ chelating ability (Kd) of 770 nM and it ispresumed to be able to respond to an expected drastic increase inintracellular calcium concentration, so that it can be presumed thatvariations of the intracellular Ca²⁺ in the control group show that theCa²⁺ pump of the cytoplasmic membrane reaches its functional limit. Inthe preparations obtained from mice treated with 30 mg/kg or 100 mg/kgof β-NMN or the preparations obtained from mice treated with from 0.01mg/kg to 1.0 mg/kg of Specimen 1 (TND1128), variations of thecytoplasmic calcium concentration at each administration show similarrecovery. As can be observed from FIG. 35 and FIG. 36, however, whenthose values were quantified by a method similar to that formitochondria, a significant difference was not observed atconcentrations other than that of the group administered with 10 mg/kgof β-NMN.

Effecting Manner of Both Drugs in Terms of Effective Concentration

Both drugs show a similar level of mitochondrial Ca²⁺ concentrationsuppressing effect. Judging from the effective concentration, the effectof Specimen 1 (TND1128) was 100 times stronger. Non-Patent Document 6has reported that NAD⁺ reached a peak 30 minutes after administration ofβ-NMN and the blood level of the administered NMN decreasescorrespondingly, suggesting the possibility of the administered NMNbecoming a matrix for NAD⁺ biosynthesis. The effective amount ofSpecimen 1 (TND1128) used in the present experiment was as trace as 1.0mg/kg or less and it cannot be considered as a raw material for thebiosynthesis, different from β-NMN. Even if the biosynthesis amount ofNAD⁺ increases in an oxidative energy acquiring process of mitochondriafinally, it should be considered that a significant mitochondrialfunction stabilizing effect observed 24 hours after the administrationowes to an increase in NAD⁺ caused by expression promotion of a sirtuingene group. Since in the present research, 24-hr pretreatment with β-NMNinduces significant mitochondrial protective function, it should beconsidered that the effect of β-NMN does not result simply from supplyof it as a a substrate for NAD⁺ but is mediated by the effect on thesirtuin gene group.

On the other hand, β-NMN and Specimen 1 (TND1128) each showed nosignificant effect on a drastic increase in cytoplasmic Ca²⁺concentration caused by 80K-ACSF. Effects of 100 mg/kg of β-NMN and 1mg/kg of Specimen 1 (TND1128) on the variations of a mitochondrial Ca²⁺concentration shown in FIG. 29 and FIG. 30 and effects of β-NMN andSpecimen 1 (TND1128) on the variations of a cytoplasmic Ca²⁺concentration shown in FIG. 33 and FIG. 34 have revealed thatirrespective of severe stimulation of 80K-ACSF, the preparations canmaintain a markedly stable physiological response. If this can bereproduced in the human brain, a strong brain protective effect can beexpected.

Pharmacological effects of synthetic sirtuin activating drugs such asresveratrol, a component of red wine, and 5RT1720, a derivative thereof,have already been reported (Non-Patent Document 8), but there is notreport actually referring to the effectiveness on the mitochondrialfunction of subcutaneously administered mouse brain as in the presentexperiment.

As is apparent from the above description, Specimen 1 (TND1128) is ahighly hydrophobic and stable compound, which is a large advantage overβ-NMN.

By making use of this advantage, this compound can be prepared into anexternal medicine and can be used for activating the damaged hair root,thereby treating silver hair or baldness or for rejuvenating flabby skincells. Further, the compound is also expected to have an effect on thebrain after being percutaneously absorbed.

The above experimental results show that the 5-deazaflavin compoundsrepresented by the formulas (I) to (IV) activate intracellular ATPproduction.

1. Use of a 5-deazaflavin compound represented by the following formula(I):

(wherein, R₁ represents a hydrogen atom, an alkyl group, ahalogen-substituted alkyl group, a carboxy-substituted alkyl group, or aphenyl group, R₂ represents an alkyl group, a cycloalkyl group, aphenyl-substituted lower alkyl group, a phenyl group, a phenyl groupsubstituted by one of a halogen atom, a lower alkyl group, or a loweralkoxy group, or a lower alkyl disubstituted phenyl group, and R₃ and R₄each represent a hydrogen atom, a lower alkyl group, a halogen atom, ahydroxyl group, a nitro group, a cyano group, a lower alkoxy group, aphenyl-substituted lower alkoxy, a lower alkylamino group, aphenyl-substituted lower alkylamino group, or a lower alkylsulfonylgroup) for activating intracellular ATP production.
 2. Use of apyridodipyrimidine compound represented by the following formula (II):

(wherein, R₁ and R3 each represent a hydrogen atom, an alkyl group, ahalogen-substituted alkyl group, a carboxy-substituted alkyl group, aphenyl group, a phenyl group substituted by one of a halogen atom, alower alkyl group, or a lower alkoxy group, or a lower alkyldisubstituted phenyl group and R₂ represents an alkyl group, acycloalkyl group, a phenyl-substituted lower alkyl group, a phenylgroup, a phenyl group substituted by one of a halogen atom, a loweralkyl group, or a lower alkoxy group, or a lower alkyl disubstitutedphenyl group) for activating intracellular ATP production.
 3. Use of adeazaflavino-testosterone compound represented by the following formula(III):

(wherein, R₁ represents a hydrogen atom, an alkyl group, ahalogen-substituted alkyl group, a carboxy-substituted alkyl group, aphenyl group, or a phenyl group substituted by one of a halogen atom, alower alkyl group, or a lower alkoxy group and R₂ represents an alkylgroup, a cycloalkyl group, a phenyl-substituted lower alkyl group, aphenyl group, a phenyl group substituted by one of a halogen atom, alower alkyl group, or a lower alkoxy group, or a lower alkyldisubstituted phenyl group) for activating intracellular ATP production.4. Use of a deazaflavino-cholesterol compound represented by thefollowing formula (IV):

(wherein, R₁ represents a hydrogen atom, an alkyl group, ahalogen-substituted alkyl group, a carboxy-substituted alkyl group, aphenyl group, or a phenyl group substituted by one of a halogen atom, alower alkyl group, or a lower alkoxy group and R₂ represents an alkylgroup, a cycloalkyl group, a phenyl-substituted lower alkyl group, aphenyl group, a phenyl group substituted by one of a halogen atom, alower alkyl group, or a lower alkoxy group, or a lower alkyldisubstituted phenyl group) for activating intracellular ATP production.